Lasers in Surgery and Medicine 12500-505 (1992)

Changes in Type I Collagen Following Laser Welding Lawrence S. Bass, MD, Nader Moazami, MD, Joanne Pocsidio, BA, Mehmet C. 0 2 , MD, Paul LoGerfo, MD, and Michael R. Treat, MD lnstitufe of Reconstructive Plastic Surgery, New York University Medical Center (L.S.B.), New York City 10016, and Department of Surgery, Columbia-Presbyterian Medical Center (N.M., J.P, M.C.O., F! L., M.R. T), New York City 10032

Selection of ideal laser parameters for tissue welding is inhibited by poor understanding of the mechanism. We investigated structural changes in collagen molecules extracted from rat tail tendon (>go%type I collagen) after tissue welding using an 808 nm diode laser and indocyanine green dye applied to the weld site. Mobility patterns on SDS-PAGE were identical in the lasered and untreated tendon extracts with urea or acetic acid. Pepsin incubation after acetic acid extraction revealed a reduction of collagen alpha and beta bands in lasered compared with untreated specimens. Circular dichroism studies of rat tail tendon showed absence of helical structure in collagen from lasered tendon. No evidence for covalent bonding was present in laser-treated tissues. Collagen molecules are denatured by the laser wavelength and parameters used in this study. N o significant amount of helical structure is regenerated on cooling. We conclude that noncovalent interactions between denatured collagen molecules may be responsible for the creation of tissue welding. 0 1992 Wiley-Liss, Inc.

Key words: diode, electrophoresis, mechanism, tendon, bond, soldering

INTRODUCTION

Laser welding offers several potential advantages over suture anastomosis or closure. These include faster healing [I], less inflammatory response [1,21, higher threshold for infection, a better flow surface [3J, greater ease and speed [3], and the ability of the weld site to grow [4]. Laser tissue welding has been evaluated in a variety of experimental models involving blood vessels 11,41, skin [51, nerve 161, and bile ducts 171. A wide range of laser wavelengths has been used, including CO, (10.60 Fm) [4,61, argon ion (0.5140.488 p m ) [1,7,81, Nd:YAG (1.06 and 1.32 pm) [5,9], thulium-holmium-chromium:YAG (2.15 pm) [3,7], and 0.808 pm diode [2] lasers. Despite the fact that investigational clinical application of laser tissue welding is currently underway, the actual mechanism for production Of the tissue is not known* unknown is the set of laser parameters or even tissue temperature. An understanding of the mechanism of laser 0 1992 Wiley-Liss, Inc.

welding would foster selection of laser parameters that maximize the welding effect within the limits of acceptable thermal damage t o the target tissue. The initial step in the formation of the weld is tissue heating as a result of absorption of laser light by tissue pigments or water. This heating is hypothesized t o produce structural changes in extracellular matrix proteins. These changes may involve uncoiling of the secondary or tertiary molecular structure or actual cleavage of the molecules. Following these structural changes, electrostatic or even covalent bonding may then occur as the final step in the formation of the weld. Because of its prominent role as a structural protein, collagen is likely to be involved in the welding Accepted for publication May 20, 1992. Address reprint requests to Dr. Lawrence S. Bass, Institute of Reconstructive Plastic Surgery, NYU Medical Center, 550 First Avenue, New York, NY 10016.

Structural Changes in Collagen 501 process. Using electrophoretic mobility patterns size (2 mm), which corresponds to a power density and circular dichroism measurements, we inves- of 14 W/cm2. Each area of the specimen was irratigated the structural changes in extracted type I diated until maximum shrinkage was produced without any signs of charring. Average duration collagen molecules after laser welding. of exposure was 30 sec for each specimen. This technique follows our previously reported method MATERIALS AND METHODS for laser tissue welding [2,81. Eleven sets of tenTissue Model don specimens were prepared for biochemical Rat tail tendon is composed almost exclu- analysis. Additional apecimens were prepared for tensively of type I collagen [lo], constituting a relatively simple, homogenous, and well-documented sile strength measurement of these end-to-end tissue system in which molecular alterations in tendon fascicle welds. Three sets of specimens type I collagen following laser exposure can be were prepared utilizing as the welding end point: (1) maximum tissue shrinkage, (2) onset of tissue clearly characterized. shrinkage, and (3) desiccation and shrinkage Laser System without smoking or charring. Laser treatments were performed with a System 2000 diode laser (808 -+ 1 nm) (Spectra- Tensile Strength Testing Samples were fixed in place on a 10 kg maxPhysics, Mountain View, CA). This unit delivers monochromatic, coherent light via a 100 p,m silica imum load cell of a T-10 Tensometer (Monsanto, fiber. This is coupled with a focusing optic to pro- Akron, OH) immediately after welding. The duce a 2 mm spot size. Output power was mea- length and thickness of each weld were measured sured with a Model 201 laser power meter (Coher- using a micrometer. The tendons were elongated ent Science Division, Palo Alto, CAI. Maximum at a constant rate of 10 c d m i n until separation of power output for this laser unit (450 mW) was the weld occurred. Peak strength recorded prior to separation was taken as the tensile strength, reutilized in production of all specimens. ported in g/cm2 after adjustment for weld crosslndocyanine Green Dye Preparation sectional area. Indocyanine green dye (ICG, Sigma, St. Louis, MO) was prepared from a mixture of 20 mg Collagen Extraction The laser-treated and untreated samples ICG and 5 ml 0.9% sodium chloride solution adjusted to a pH of 4.5-7. The resulting dark green were extracted with 10 ml of one of the following solution exhibits maximum absorbance at 805 solutions: (1)0.5 M acetic acid, (2) 8 M urea, (3) nm, acting as a topical chromophore to concen- 0.5 M acetic acid, pepsin (enzymexubstrate ratio trate the conversion of laser light to heat in the l : l O ) , (4) 0.5 M acetic acid, 2% ethanol (circular target tissue [2]. The peak absorbance of this dichroism analysis only), and (5) 0.5 M acetic chromophore is well matched to the wavelength of acid, cyanogen bromide (enzyme:substrate ratio laser light used. At the power densities used in 1:10). Protease inhibitors, 1 mM phenylmethylthis study, no tissue effect is observed in un- sulfonly fluoride (PMSF), and 5 mM ethylene ditreated tissues. Tissue stained with the chro- aminetetraacetate (EDTA), were added to the mophore absorbs the laser light heavily with non-enzymatic extractions (Groups 1,2, and 4). All samples were extracted overnight by stirring clearly observable tissue effects. at 4" C, followed by centrifugation at 3,000 rpm Operative Technique for 5 min. The pellet was saved at -20" C for gel The tail tendon from 3-5-month-old Sprague- electrophoresis. The supernatant was extensively Dawley rats was excised immediately after sacri- dialyzed against water for 24 hr, lyophilized, and fice. Individual tendon fascicles were dissected stored at -20" C. free and divided into 5 mg segments. A 0.5 ml aliquot of ICG was uniformly applied to each spec- SDS-Polyacrylamide Gel Electrophoresis Specimens were studied using sodium dodeimen. During application of laser light, the focus- cylsulfate (SDS) 10% polyacrylamide gel electroing optic was kept a fixed distance from the tissue phoresis (PAGE) [ll]. Extracts from each laser being treated while exposing all tissue in a "paint specimen and control underwent repeated analybrush" fashion. This resulted in a uniform spot sis in multiple gels and multiple lanes within a

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gel. Each lane in a gel was loaded with identical amounts of lyophilized protein from the extraction fractions being tested. After completion of electrophoresis, the gel was stained with a 0.1% coomassie blue solution and dried on a filter paper backing. The bands were quantified by measuring the optical density using the 300A Computing Densitometer (Molecular Dynamics, Sunnyvale, CA). Circular Dichroism Studies

In addition to the samples that were prepared as described above, 5 mg of tendon was extracted with 0.5 M acetic acid and 2% ethanol, and heated at 60" C for 2 hr to completely denature all collagen in the specimen. Prior t o analysis, all samples were dissolved in a 0.1 mM sodium phosphate solution (pH 6.0) to give a concentration of 0.05 mg/ml. Circular dichroism (CD) measurements were carried out on a 5-600 spectropolarimeter (Japan Spectroscopic Co., Tokyo, Japan) at 37" C immediately after reconstitution. The resulting tracings were obtained from signal averaging of five scans of each specimen. RESULTS Tensile Strength

Mean tensile strength of tendons in Group 1 (maximum shrinkage) was 160 k 44 g/cm2 (n = 7). Welds in the other two groups produced no measureable weld strength on the tensometer. Grossly, these welds were observed to be easily disrupted with minimal tissue manipulation. Electrophoresis

Electrophoretic migration patterns for lasertreated and nontreated rat tail tendons extracted in urea were identical (Fig. 1, Lanes 3,4) except for a slight widening of laser treated bands. Material not solubilized by urea extraction also had identical patterns. The characteristic bands seen in type I collagen are present including alpha 1 and 2 and Beta 1,l and 1,2. Urea denatures the protein strands in each fibril. Acetic acid extracts collagen molecules without denaturing the helical structure [12]. Treatment of acetic acid extracted tendon with pepsin cleaves the nonhelical portions of collagen molecules at the pepsin recognition sites. Acetic-acid extracted, pepsintreated rat tail tendon preserves a similar pattern t o non-pepsin-treated collagen with alpha and beta chains easily recognized (Fig. 1, lanes 2,6). Lasered rat tail tendon displays a loss of charac-

Fig. 1. SDS PAGE of lasered (L) and nonlasered rat tail tendon, pepsin digestion. Lane 1: lasered, acetic acid (A) extraction. Lane 2: nonlasered, acetic acid extraction. Lane 3: lasered, urea (U) extraction. Lane 4 nonlasered, urea extraction. Lane 5 lasered, acetic acid extraction, pepsin (P) incubation. Lane 6 nonlasered acetic acid extraction, pepsin incubation. No difference is seen between lasered and nonlasered specimens on urea extraction. Alpha and beta bands are markedly reduced after pepsin incubation in lasered specimens.

teristic collagen bands when treated with pepsin (Fig. 1 lanes 1,9. This suggests extensive loss of helical structure, allowing pepsin to cleave the protein chains at multiple sites. Material not solubilized by urea extraction also had identical patterns in lasered and untreated groups. Cyanogen bromide treatment results in an identical pattern in lasered and untreated specimens with near total loss of alpha and beta collagen bands (Fig. 2, lanes 1,2). This represents significant cleavage of the collagen molecules in both types of specimens. The mobility patterns provide no support for the presence of novel covalent bonds. Optical Densitometry

There is no significant difference between laser-treated and non-treated rat tails in the density of alpha and beta bands of acetic acid or urea treated extracts. The density of bands compared between treatment and control lanes is not significantly different nor is the fraction of total density within each lane. However, there is significant loss of density in alpha and beta bands in pepsin treated laser samples compared with non-lasered samples (Table 1).

Structural Changes in Collagen

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TABLE 1. Optical Density Ratio of Lasered to Unlasered Collagen Bands on SDS PAGE of Rat Tail Tendon (Fig. 1) Sample"

Alpha 2 0.84 1.0 0.0

Alpha 1 0.97 1.1 0.0

U A AP

Beta 1,2 1.11 0.93 0.0

Beta 1,l 1.07 0.87 0.0

"U = urea extraction; A = acetic acid extraction; AP acid extraction, pepsin incubation. 101

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Fig. 2. SDS PAGE of lasered (L) and nonlasered rat tail tendon, cyanogen bromide digestion. Lane 1: non-lasered, acetic acid (A) extraction, cyanogen bromide (C)incubation. Lane 2 lasered, acetic acid extraction, cyanogen bromide incubation. Lane 3: nonlasered, acetic acid extraction. Lane 4: lasered, acetic acid extraction. Patterns between lasered and nonlasered specimens were identical. Some residual undigested collagen is visible in cyanogen bromide-treated specimens, matching bands in lanes 3 and 4.

Circular Dichroism

Native collagen extracted from rat tail tendon displays the characteristic negative ellipticity at 197 nm and positive ellipticity at 222 nm (Fig. 3) [131. In contrast, heat-denatured collagen possessed a negative ellipticity at 222 nm. Lasertreated collagen demonstrates a circular dichroism curve similar t o that for heat-denatured collagen. The area under the curve peak at 222 nm was approximately zero. This indicates near total absence of helical collagen. DISCUSSION

Schober et al. [9] demonstrated a loss of periodicity, increased caliber, and interdigitation of collagen fibrils in rat carotid arteries anasto-

Fig. 3. Ellipticity (mdeg cm2/dmol) of collagen purified from rat tail tendon. Helical structure is demonstrated in native collagen by the area under the curve peaking at 222 nm. Heat-denatured collagen is devoid of helicity. Laser-treated collagen possesses minimal helical structure.

mosed with the 1.32 pm Nd:YAG laser. The molecular mechanism responsible for these ultrastructural changes was not identified. White et al. [141 reported loss of striation and swelling of collagen fibrils to 100-400 pm (normally 70 pm) in canine arteriovenous fistulas created with the argon laser. The same authors demonstrated an increased amount of high molecular weight proteins in guanidine extracts of laser-treated skin compared with untreated controls as well as an increased amount of low molecular weight protein in treated blood vessels compared with control specimens 1151. Whereas this suggests that covalent cross-linking (in one tissue system) may result from laser irradiation, the presence of other concurrent potential mechanisms of welding was not evaluated. Therefore, it is not clear whether these covalent bonds are necessary for tissue welding or are an incidental finding. The ultrastructural changes noted in the collagen fibrils could be explained either by covalent cross-linking or by protein denaturation with noncovalent bonding in a helical or aggregate form. In our study we examined changes in rat tail

Bass et al. tendon, a nearly pure type I collagen preparation. system. In this system, our results indicate that: Our results support the conclusion that noncova- (1) novel covalent bonds are not occurring in any lent bonding between denatured collagen strands appreciable amount (to the limit of detection and can create a laser weld. No evidence for covalent in the molecular weight range evaluated on gel cross-linking could be seen in any of the gels pre- electrophoresis), (2) the type I collagen is denapared. There were no unanticipated high molecu- tured (loss of secondary structure), (3) no apprelar weight bands in the running gel or stacking ciable amount of helicity is regenerated in type I gel of the laser-treated specimens. Covalent bonds collagen on cooling, and (4) the necessary condishould not be disrupted by any of the nonenzy- tions exist for production of strong noncovalent matic treatments in the extraction or analysis bonding after laser exposure. In general, covalent process [12,16]. Urea extraction completely dena- or disulfide bonding may contribute to weld tures the protein molecules, whereas acetic acid strength in other circumstances, but adequate extraction does not, leaving helical collagen in- welding can be generated in their absence. tact. Pepsin will cleave nonhelical collagen, but By understanding the mechanisms responsileaves helical collagen intact. Since acetic acid ble for the generation of structural strength in does not denature collagen, pepsin cleavage of la- laser welding, we hope to understand the consered collagen implies denaturing of protein sec- ditions necessary for its creation. Our results ondary to heating during irradiation. suggest that tissue welding can be created at temWe also found that lasered collagen does not peratures that result in protein denaturation. regenerate a significant amount of helical struc- Therefore, if peak temperature and duration of ture on cooling. Such an effect is unlikely for sev- exposure are carefully controlled, laser parameeral reasons [13]. Reannealing of collagen occurs ters could be selected that would create tissue only with difficulty and imperfectly in vitro. In welding with a narrow zone of cell death. addition, there is a large disparity between the time required for reannealing and the duration of laser irradiation during welding. The denatured REFERENCES randomly oriented collagen thus has the opportu- 1. White RA, Kopchok G, Donayre C, White G, Lyons R, nity to noncovalently aggregate in a variety of Fujitani R, Klein SR, Uitto J. Argon laser-welded arteriovenous anastomosis. J Vasc Surg 1987; 6:447-453. disordered forms. The production of strong hydrophobic interstrand interactions in nonhelical do- 2. Oz MC, Chuck RS, Johnson JP, Parangi S, Bass LS, Nowygrod R, Treat MR. Indocyanine green dye-enhanced mains of collagen molecules has been postulated welding with a diode laser. Surg Forum 1989; 40:316by others [13]. 318. There are some differences in the collagen in 3. Bass LS, Treat MR, Dzakonski C, Trokel SL. Sutureless microvascular anastomosis using the THC:YAG laser: A other body tissues that may limit the scope of preliminary report. Microsurgery 1989; 10:189-193. these results. Native covalent-linking between 4. Frazier OH, Painvin GA, Morris JR, Thomsen S, Neblett collagen strands increases with age [lo]. In addiCR. Laser-assisted microvascular anastomoses: Angiotion to type I collagen, blood vessels contain type graphic and anatomopathologic studies on growing miI11 and V collagen, which are relatively hydroxycrovascular anastomoses: Preliminary report. Surgery 1985; 97585-589. proline rich [161. These hydroxyproline residues stablize the helical conformation of collagen mol- 5. Abergel RP, Lyons RF, White RA, Lask G, Matsuoka LY, Dwyer RM, Uitto J. Skin closure by Nd:YAG laser weldecules, resulting in greater temperature stability ing. J Am Acad Dermatol 1986; 14810-814. (higher melting temperature) [171. The amino 6. Maragh H, Hawn RS, Gould JD, Terzis JK. Is laser nerve acid side-chains in type I collagen do not allow for repair comparable to microsuture coaptation? J Reconstruct Microsurg 1988;4:189-195. disulfide bond formation, which some have suggested may play a role in tissue welding [18]. The 7. Oz MC, Bass LS, Popp HW, Chuck RS, Johnson JP, Trokel SL, Treat MR. In vitro comparison of thuliumpresent study demonstrates that tissue welding holmium-chromium:YAG and argon ion lasers for weldcan take place in the absence of disulfide bonding. ing biliary tissue. Lasers Surg Med 1989; 9:248-253. The nature of tissue effects generated may 8. Chuck RS, Oz MC, Delohery TM, Johnson JP, Bass LS, Nowygrod R, Treat MR. Dye-enhanced laser tissue weldvary significantly with the laser wavelength, paing. Lasers Surg Med 1989; 9:471-477. rameters, and tissue model employed. Different or 9. Schober R, Ulrich F, Sander T, Durselen H, Hessel S. multiple mechanisms may be responsible for the Laser-induced alteration of collagen substructure allows welding effect in different systems. With the pamicrosurgical tissue welding. Science 1986; 232:1421rameters and techniques noted above, tissue 1422. welding can be created in a pure type I collagen 10. Tanaka S, Avigad G, Eikenberry EF, Brodsky B. Isola504

Structural Changes in Collagen tion and partial characterization of collagen chains dimerized by sugar-derived cross-links. J Biol Chem 1988; 263:17650-17657. 11. Studier FW. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol. Biol 1973; 79:237-248. 12. Miller EJ. Chemistry of collagens and their distribution. In: Piez KA, Reddi AH, eds. “Extracellular Matrix Biochemistry.’’ New York: Elsevier, 1984; p. 58. 13. Schmid TM, Linsenmayer TF. Denaturation-renaturation properties of two molecular forms of short-chain cartilage collagen. Biochemistry 1984; 23553-558. 14. White RA, Kopchok GE, White GH, Klein SR, Uitto J . Laser vascular anastomotic welding. In White RA, Grundfest WS, eds. “Lasers in Cardiovascular Disease.”

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Chicago: Year Book Medical Publishers, 1987, pp 103117. Murray LW, Su L, Kopchok GE, White RA. Crosslinking of extracellular matrix proteins: A preliminary report on a possilbe mechanism of argon laser welding. Lasers Surg Med 1989; 9:490-496. Miller EJ, Rhodes RK. Preparation and characterization of the different types of collagen. Methods Enzymol 1982; 82:33-64. Gelman RA, Blackwell J, Kefalides NA, Tomichek E. Thermal stability of basement membrane collagen. Biochim Biophys Acta 1976; 427:492-496. Helmsworth TF, Wright CB, Scheffter SM, Schlemm DJ, Keller SJ. Molecular surgery of the basement membrane by the argon laser. Lasers Surg Med 1990; 10:576-583.

Changes in type I collagen following laser welding.

Selection of ideal laser parameters for tissue welding is inhibited by poor understanding of the mechanism. We investigated structural changes in coll...
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